Tumor cell metastasis is a complex multistep process involving homotypic and heterotypic interactions among tumor cells and host cells (i.e., platelets, endothelial cells, etc.), in addition to tumor cell interactions with the extracellular matrix. These interactions are mediated by a variety of cell surface receptors including cadherins, selectins, and integrins. Tang, D. G. et al., Invasion Metastasis 95:109-122 (1994).
Integrins are heterotypic adhesion receptors involved in cell-cell and cell-matrix interactions. At least 15 alpha (.alpha.) and 8 beta (.beta.) subunits have been identified that can pair differently to form more than 20 receptors. Hynes, R. O., Cell 69:11-25 (1992); Schwartz, M. A., et al., Ann. Rev. Cell Dev. Biol. 1 1:549-599 (1995). Most cDNAs coding for the known integrins have been cloned and sequenced, and their genes localized to chromosomes. (Block, K. L. et al., Stem Cells 13:135-145 (1995).
The platelet integrin .alpha.IIb.beta.3 (also known as GP IIb-IIIa) is the prototypical integrin receptor and its structure has been studied in great detail. Calvete, J. J., Throm. Haemostasis 72:1-15 (1994). Integrins generally contain a large extracellular domain formed by the .alpha.(.about.1,000 amino acids) and .beta.(.about.750 amino acids) subunit, a single transmembrane segment from each subunit, and two short cytoplasmic tails, with the exception of .beta.4, whose cytoplasmic tail is more than 1,000 amino acid residues in length. Sastry, S. K. et al., Current Opin. Cell Biol. 5:819-831 (1993). The .beta. subunit is a single chain polypeptide with 4 cysteine repeats in the extracellular domain. The .alpha. subunit is a single gene product that is postranslationally cleaved into the light and heavy chain which are re-connected by a disulfide bond. The light chain of the .alpha. subunit contains the transmembrane and the cytoplasmic tail. Even though integrins were originally thought to function purely as anchor molecules, they are also known as signaling receptors. Integrin cytoplasmic tails do not have intrinsic enzymatic activity, but by recruiting and activating tyrosine (pp125FAK, pp60src), serine (PKCa, ERK, JNK, ILK) or lipid (cPLA2, PI3K, PI4P5K) kinases, they can simultaneously control multiple signaling pathways such as the MAP kinase and JAK-STAT pathways. Clark, E. A. et al., Science 268:233-2239 (1995); Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995).
Human and rat cDNAs of .alpha.IIb as well as human cDNA of .beta.3 have been cloned. Poncz, M. et al., J. Biol. Chem. 262:8476-8482 (1987); Poncz, M. et al., Blood 75:1282-1289 (1990); Fitzgerald, L. et al., J. Biol. Chem 62:3936-3939 (1987). Genes of human .alpha.IIb and .beta.3 have been localized to chromosome 17, and their structures have been determined. Heidenreich, R. et al., Biochem. 29:1232-1244 (1990); Lanza, F. et al., J. Biol. Chem. 265:18098-18103 (1990); Bray, P. F. et al., J Clin. Invest. 80:1812-1817 (1987); Sosnoski, D. M. et al., J. Clin. Invest. 81:1993-1998 (1988); Rosa, J. P. et al., Blood 72:593-600 (1988). The integrin .alpha.IIb gene is believed to be under stringent megakaryocyte specific transcriptional control whereas .beta.3 is widely expressed. Prandini, M. H. et al., J. Biol. Chem. 267:10370-10374 (1992); Calvete, J. J. et al., Throm. Haemostasis 72:1-15 (1994); Block, K. L. et al., Stem Cells 13:135-145 (1995).
Integrins are conformationally labile and they can exist in an inactive and active form. The inactive integrin recognizes ligand with low affinity while the active integrin recognizes ligand with high affinity. For example, in resting platelets .alpha.IIb.beta.3 is constitutively expressed in an inactive form, and only after platelet activation is the integrin converted to an active state which then binds plasma fibrinogen with high affinity, thereby resulting in platelet aggregation. Shattil, S. J. et al., Current Opin. Cell Biology 6:695-704 (1994). Integrins can be activated by extracellular signals such as divalent cations (Mn.sup.2+, Ca.sup.2+) or by treatment with certain activating mAbs. Schwartz, M. A. etal., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995). Such activation induces a conformational change without involving cellular metabolism, a phenomenon referred to as "outside-in" signaling. On the other hand, growth factor-mediated activation of intracellular kinases and phosphatases can result in integrin activation, a phenomenon referred to as "inside-out" signaling. Integrins therefore participate in bi-directional signaling. Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995). Several reports in the literature indicate that inside-out signaling is mediated by the cytoplasmic tail of the .alpha. subunit, and outside-in signaling by the cytoplasmic tail of the .beta. subunit. Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995); Shattil, S. J. et al., Current Opin. Cell Biology 6:695-704 (1994).
Although the relationship between integrin receptors and metastasis is variable, most integrins expressed by tumor cells are also expressed by their normal counterparts, however some tumor cells express certain integrins that are not expressed by their normal counterparts, a phenomenon referred to as "ectopic" or "abnormal" expression. For example, several groups have demonstrated the ectopic expression of .alpha.IIb.beta.3 in non-megakaryocytic cells derived from solid tumors. Chen, Y.Q. et al., J. Biol. Chem. 267:17314-17320 (1992); Trikha, M. et al., Cancer Res. 56:5071-5078 (1996); Trikha, M. et al., Cancer Res. 57:2522-2528 (1997); Boekerche, H. et al., Blood 74:658-663 (1989); Kamiyama, M. et al., Cancer Res. 53:221-223 (1993); Chiang, H. S. et al., Biochem. et Biophys. Acta 1224:506-516 (1994); Puerschel, W.Ch. et al., British J. Dermatol. 135:883-887 (1996). This receptor participates in tumor cell-platelet, -endothelial cell, and -ECM interactions. Chiang, H. S. et al., Biochim. et Biophys. Acta 1224:506-516 (1996); Tang, D. G. et al., Invasion Metastasis 95:109-122 (1994); Honn, K. V. et al., Exp. Cell Res. 201:23-32 (1992). In addition, subpopulations from melanoma tumors, which differ in their metastatic potential, demonstrate a positive correlation between .alpha.IIb.beta.3 expression and lung colony formation. Tang, D. G. et al., Intl. J. Cancer 54:338-347 (1993). Two mAbs directed to .alpha.IIb.beta.3, 10E5 and PAC-1, inhibit lung colonization of tail vein injected human prostate adenocarcinoma DU-145 cells in SCID mice. Trikha, M. et al., Prostate In Press (1997). Collectively, these findings suggest that the platelet .alpha.IIb.beta.3 integrin is ectopically expressed in non-megakaryocytic lineage tumor cells, and it participates in tumor cell metastasis. It is conceivable that as a result of transformation events transcription of the .alpha.IIb gene is elevated in some tumors which allows them to interact with the host in a platelet-type manner thereby facilitating the metastatic process. Recent studies indicate that human melanoma cells express an intracellular pool of constitutively active .alpha.IIb.beta.3, because these permeabilized cells recognize PAC-1, a unique mAb because it specifically recognizes the high affinity state of .alpha.IIb.beta.3 integrin. Shattil, S. J. et al., J. Biol. Chem. 260:11107-11114 (1995); Trikha, M. et al., Cancer Res. 57:2522-2528 (1997). Earlier reports in the literature indicate that CHO cells transfected with wt-.alpha.IIb and wt-.beta.3 cDNAs constitutively express .alpha.IIb.beta.3 in a low affinity state. Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995); O'Toole, T.E. et al., J. Cell Biol. 124:1047-1059 (1994). However, transfection with mutant .alpha.IIb constructs that either have a point mutation in the cytoplasmic tail or complete deletion of the tail, result in constitutive expression of the integrin in a high affinity state. Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599 (1995); O'Toole, T. E. et al., J. Cell Biol. 124:1047-1059 (1994); O'Toole, T. E. et al., Science 254:845-847 (1991). To date no such naturally occurring mutant constructs had been observed.